Lower alkoxy inosine-5&#39;-phosphates and salts thereof

ABSTRACT

THE 2-(LOWER-ALKOXY) INOSINE-5&#39;&#39;-PHOSPHATES (2-METHOXY-, 2-ETHOXY-, 2-N-PROPOXY- AND 2-ISOPROPOXY-INOSINE-5&#39;&#39;PHOSPHATES) HAVE A HIGH CAPACITY FOR IMPROVING THE FLAVOR OF FOODS AND BEVERAGES. MOEROVER, THERE IS A SIGNIFICANT SYNERGISTIC ACTION THEREBETWEEN AND MONOSODIUM GULTAMATE.

United States Patent 3,644,331 LOWER ALKOXY INOSINE-5'-PHOSPHATES AND SALTS THEREOF Ryuji Marumoto, Minoo, Yoshio Yoshioka, Osaka, Hisashi Aoki, Suita, and Jun Toda, Nishinomiya, Japan, gssignors to Takeda Chemical Industries, Ltd., Osaka,

apan N0 Drawing. Filed June 26, 1968, Ser. No. 740,094 Claims priority, application Japan, June 26, 1967, 42/ 10,899 Int. Cl. C07d 51/54 US. Cl. 260-211.5 R 7 Claims ABSTRACT OF THE DISCLOSURE The 2-(lower-alkoxy)inosine-5-phosphate's (2-methoxy-, 2-ethoxy-, 2-n-propoxyand 2-isopropoxy-inosine-5'- phosphates) have a high capacity for improving the flavor of foods and beverages. Moreover, there is a significant synergistic action therebetween and monosodium glutamate.

This invention relates to a novel seasoning composition, a method for preparing the same and a method for improving or enhancing flavor of foods or beverages.

5'-purine-nucleotides such as 5-inosinic acid and 5- guanylic acid have practically been employed as chemical condiments because of their strong flavor-enhancing effect (see French Pats. Nos. 1,219,220 and 1,255,334).

It has been found by the present inventors that 2-(lower alkoxy)inosine-5'-phosphates of the formula:

9 II R no P o 011 on n wherein R stands for a lower alkoxy group having at most 3 carbon atoms, all of them being novel compounds, have a much higher ability of improving or enhancing flavor of foods or beverages than that of 5-inosinic acid.

It has further been found that there is a significant synergistic action between the 2-(lower alkoxy)inosine-5- phosphates (I) and monosodium glutamate.

It is an object of the present invention to provide a novel seasoning composition. Another object of the present invention is to provide a method for preparing the said seasoning composition. Further object of the present invention is to provide a method for improving or enhancing the flavor of food or beverages.

The 2-(lower alkoxy)inosine-5'-phosphates (I) include 2-methoxyinosine 5' phosphate, 2-ethoxyinosine-5- phosphate, 2-n-propoxyinosine 5' phosphate and 2-isopropoxyinosine-5'-pho'sphate.

These 2-(lower alkoxy)inosine-5'-phosphates are prepared, for example, by phosphorylating a corresponding 2-(lower alkoxy)inosine or 2-(lower alkoxy)-2',3-isopropylideneinosine and subsequently subjecting the resultant product to hydrolysis. As the phosphorylating agent, pyrophosphoryl tetrachloride, phosphoryl chloride or partially hydrolyzed phosphoryl chloride may most advantageously be employed. The phosphorylating reaction proceeds smoothly at a temperature between 0 and 10 C. If a reaction solvent is necessary, there can be employed phenols such as phenol, cresol, xylenol, etc.

3,644,331 Patented Feb. 22, 1972 The hydrolysis of the thus-obtained product is carried out by per se known procedure, for example by pouring the reaction mixture into water, preferably cooled water, or by weakening the acidity of the reaction mixture desirably to about pH 1 to 2 with the addition of an alkaline material such as sodium hydroxide, sodium carbonate and, if necessary, heating the mixture at a temperature between 60 and C. for 15 to 60 minutes.

The 2- (lower alkoxy)inosine or 2- (lower alkoxy) -2',3- isopropylideneinosine to be employed as the starting material can be prepared, for example, by subjecting an alkali metal salt or ammonium salt of Z-mercaptoinosine to the action of chlorine gas at 0 to 10 C. in an alcohol containing concentrated hydrochloric acid and subjecting the thus-obtained 2-chloroinosine or 2echloro-2',3'-isopropyli deneinosine prepared four the 2-chloroinosine to the action of sodium alkoxide.

For the purpose of this invention, the thus-obtained 2-(lower alkoxy)inosine-5'-phosphates can be employed in the free form or as a physiologically acceptable salt such as alkali metal salt, e.g. sodium salt, potassium salt; alkaline earth metal salt, e.g. calcium salt; ammonium salt; amine salt such as cyclohexylamine salt.

The respective threshold values of the 2-(lower alkoxy) inosine-5-phosphates (1) determined by the forcedchoice method of limits described in American Journal of Psychology, vol. 69, pp. 672 to 673, are as follows:

Threshold value,

Compound: percent in water Disodium 2-methoxyinosine-5'-phosphate 0.0141 Disodium 2-ethoxyinosine-5-phosphate 0.0077 Disodium 2-n-propoxyinosine-5-phosphate 0.0119 Disodium 2-isopropoxyinosine-5'-phosphate 0.0084

As clearly shown in the above table, the respective threshold values of the 2-(lower alkoxy) inosine-5'-phosphates (I) are far lower than that of disodium-5'-inosinate per se, which means that these compounds can be detected at a lower concentration than 5'-inosinic acid. Further, as mentioned hereinbefore, when the 2-(lower alkoxy)- inosine-5'-phosphates are employed in combination with monosodium glutamate, a remarkable synergistic action is observed between these compounds and the monosodium glutamate. That is, as clearly shown in tests described hereinafter in the co-presence of monosodium glutamate, the flavor-enhancing eifect of these compounds is as much as about 5 times as strong as that of 5'-inosinic acid per se.

It is also known that 2-methylthioinosine-5'-phosphate and 2-ethylthioinosine-5'-phosphate are possessed of a flavoring effect stronger than that of 5'-inosinic acid per se (see French Patent No. 1,463,442). The 2-(lower alkoxy)inosine-5'-phosphates (I) of the present invention are more stable and less toxic than these known 5-inosinic acid derivatives.

The 2-(lower alkoxy)inosine-5'-phosphates (I) can be added to foods or beverages, in the solid state or in a liquid state, i.e. dissolved in water or hydrophilic palatable and edible organic solvent, eg. alcohol. In improving or enhancing the flavor of foods or beverages, the objective foods or beverages are mixed with a 2-(lower alkoxy)inosine-5'-phosphate (I), or are impregnated with a solution of a 2-(lower alkoxy)inosine-5-phosphate, or sprinkled over with the solution. The addition of a 2- (lower alkoxy)inosine-5'-phosphate is carried out during or after the preparation of the foods or beverages.

Foods or beverages to be seasoned include, for example, fermented foods such as bean paste (miso), soy sauce, vinegar, sake; paste such as ham paste, sausage, steamed fish paste (kamaboko or chikuwa); meats such as whale meat, poultry meat, pork, beef; noodles such as macaroni;

milk and its processed matters such as cows milk, condensed milk, cheese; processed vegetables such as tomato juice, canned spinach; cooked foods such as soups, stews.

In most cases, it is preferable to employ the 2-(lower alkoxy)inosine-'-phosphates together with monosodium glutamate and, if desired, another chemical condiment such as disodium 5-guanylate or sodium succinate. The ratio of the 2-(1ower alkoxy)inosine-5'-phosphates relative to the monosodium glutamate is advantageously about 1/500 to 1/5.

The preparation of the seasoning composition comprising the 2-(lower alkoxy)inosine-5-phosphate (I) and monosodium glutamate can be carried out by simply mixing, or by preparing a nucleus of either the 2- (lower alkoXy)inosine-5'-phosphate (I) or the monosodium glutamate and successively adhering other components to the nucleus. If desired, a composition of the 2-(lower alkoxy) inosine-5'-phosphate (I) and monosodium glutamate, irrespective of whether it is powdery or grandular, can be coated with a known coating agent. As the coating agent there may be used aliphatic acid esters of sugars, e.g. soucrose fatty acid ester, gelatin, casein, edible waxes, stearic acid, vegetable proteins, monoglycerides, etc.

The most effective amount of the 2-(lower alkoxy) inosine-5'-phosphate to be employed varies with kinds of foods or beverages, but generally, about 0.0005 to 0.03% relative to foods beverages is advantageous.

In the following Tests and Examples, percentages are by weight; parts by volume bear the same relationship to parts by weight as do milliliters to grams; two-sample directional test, constant method and probit analysis employed in the following Tests are those described on pp. 330-332 of Principles of Sensory Evaluation of Food published by Academic Press, New York and London, in 1965, pp. 118-141 of Psychometric Methods, 2nd. edition published by McGraw-Hill Book Company, Inc., New Work, and Probit analysis, a statistical treat ment of the sigmoid respense curve published by Cambridge Univ. Press in 1952, respectively.

Test 1 Sample A.Aqueous solution containing 0.8% of sodium chloride plus 0.03% of disodium Z-methyoxyinosine- 5 '-phosphate Sample B.Aqueous solution containing 0.8% of sodium chloride plus 0.03% disodium 5-in0sinate.

Two-sample directed test carried out between Sample A and Sample B, and 19 out of 20 persons judged the flavor of Sample A stronger than that of Sample B (significant at 0.1% level).

Test 2 Sample A.Aqueous solution containing 1% of soy sauce, 0.6% of sodium chloride plus 0.001% of disodium 2-methoxyionsine-5'-phosphate Sample B.Aqeous solution of the same composition as Sample A except that 0.001% of disodium Z-methoxyinosine-5-phosphate is replaced by the same amount of disodium 5-inosinate.

Two-sample directional tests were carried out between Sample A and Sample B, and 16 out of 20 persons judged the flavor of Sample A stronger than that of Sample B (significant at 1% level).

Test 3 Sample A.Aqueous solution containing 0.8% of sodium chloride and 0.03% of disodium 2-ethoxyinosine- 5'-phosphate.

Sample B.Aqueous solution containing 0.8% of sodium chloride and 0.03% of disodium 5-inosinate.

Two-sample directional tests were carried out between Sample A and Sample B, and 19 out of 20 persons judged the flavor of Sample A stronger than that of Sample B (significant at 0.1% level).

Test 4- 7 Sample A.Aqueous solution containing 1% of soy sauce, 0.6% of sodium chloride and 0.01% of disodium 2-ethoxyinosine-5'-phosphate.

Sample B.Aqueous solution of the same composition as Sample A except that 0.01% of disodium 2-ethoxyinosine-5-phosphate is replaced by the same amount of disodium 5'-inosinate.

Two-sample directional tests were carried out between Sample A and Sample B, and 17 out of 20 persons judged the flavor of Sample A stronger than that of Sample B (significant at 1% level).

To compare quantitatively the flavor-enhancing eifect of a specified 2-(lower alkoxy)inosine-5'-phosphate with that of disodium 5'-inosinate, further tests as shown in Tests 5 to 7 were carried out according to the constant methods (panel: 50 members) in which the Sample A is a standard sample and the Samples 1 to- 5 are variable ones. The concentration of disodium 5-inosinate equivalent to Sample A in flavor-enhancing effect was determined by the application of the probit analysis to the resulting data shown in Tables 1 to 3.

Samples A and 5 8 (16%) 1 Those who judged the flavor of Sample A stronger.

The probit analysis as to the results in Table 1 revealed that 0.00025 of disodium 2-methoxyinosine-5-phosphate is equivalent to 0.00092% of disodium 5-inosinate in a flavor-enhancing effect and, therefore, that the flavorenhancing effect of disodium Z-methoxyinosine-S-phos phate is 3.7 times as strong as that of disodium 5- inosinate.

TEST 0 Disodium Sodium Mouosodium Z-ethoxychloride, glutamate, iuosine-5- Samples percent percent phosphate,

percent Disodium 5 iuosinate,

percent TABLE 2 Comparison between Number of persons 1 Samples A and 1 42 (84%) Samples A and 2 38 (76%) Samples A and 3 34 (68%) Samples A and 4 22 (44%) Samples A and 5 12 (24%) 1 Those who judged the flavor 01 Sample A stronger.

The probit analysis as to the results in Table 2 revealed that 0.00025 of 2-ethoxyinosine-5-phosphate is equivalent to 0.00122% of disodium 5-inosinate in flavor-enhancing effect and, therefore, that the flavor-enhancing effect of disodium 2-ethoxyinosine-5-phosphate is 4.9 times as strong as that of disodium 5-inosinate.

TEST 7 1 )is0dium 2- rsopropoxy- Sodium Monosodiurn inosine-5- chloride, glutamate, phosphate, Samples percent percent percent Disodium 5 inosinate, percent 0. s 0. 100 0. 00055 0. s 0. 100 0. 00074: 0. s 0. 100 0. 00100 0. s 0. 100 0. 00135 8 0.100 0. 00182 TABLE 3 Number of Comparison between-- persons 1 Samples A and 1 40 (80%) Samples A and 2 32 (64%) Samples A and 3 34 (68%) Samples A and 4 24 (48%) Samples A and 5 8 (16%) 1 Those who judged the flavor of Sample A stronger.

The profit analysis as to the results in Table 3 revealed that 0.0002S% of disodium 2-isopropoXyinosine-5-phosphate is equivalent to 0.00113% of disodium 5-inosinate in flavor-enhancing effect and, therefore, that the flavorenhancing effect of 2-isopropoxyinosine-5-phosphate is 4.5 times as strong as that of disodium 5'-inosinate.

EXAMPLE 1 23 parts by weight of Z-methoxyinosine is dissolved in 1000 parts by volume of metacresol. Into the resultant solution is added dropwise with vigorous stirring 80 parts by volume of pyrophosphoryl chloride at 5 to 10 C. The mixture is kept standing with stirring at the same temperature for 3 hours. The reaction mixture is poured into 10,000 parts by volume of ice water and the Whole mixture is shaken with 10,000 parts by volume of diethyl ether to transfer the metacresol into the resulting diethyl ether layer.

The aqueous layer is adjusted to pH 7 with 1 N-sodium hydroxide solution and then is concentrated under reduced pressure to remove diethyl ether still existing in it. The aqueous layer is adjusted to pH 1 with 1 N hydrochloric acid and is allowed to pass through a column packed with 500 parts by weight of activated charcoal. The column is washed with water and is subjected to elution with 4000 parts by volume of a mixture of ethyl alcohol, n-butyl alcohol, 28% aqueous ammonia and water (45 :522248 by volume). The eluate is concentrated to 500 parts by volume. The concentrated solution is allowed to pass through a column packed with 1000 parts by volume of cation exchange resin (Na+ type of Amberlite 1R-120). The eflluent is concentrated under reduced pressure to give 25 parts by weight of residue. The residue is dissolved in 100 parts by volume of Water. The solution is kept standing in a cool place for 12 hours to give crystals of disodium Z-methoxyinosine-S-phosphate. The crystals are collected from the solution. To the solution is added 200 parts by volume of ethyl alcohol to give crystals of disodium 2-methylinosine-5- phosphate. The total yield of thus-obtained 2-methoxyinosine-5'-phosphate is 15 parts by Weight.

This product gives a single ultraviolet absorption figure in paper electrophoresis (0.05 M borate buffer, pH

9.2), the absorption A533 244 my (6 104.50

Elementary analysis.-Calculated for C 1H13O9N PNa2 (percent): C, 28.95; H, 3.71; N, 12.25. Found (percent): C, 28.67; H, 4.46; N, 12.15.

EXAMPLE 2 (A) 0.9075 part by weight of 2-chloroinosine is suspended in 50 parts by volume of acetone. To the suspension is added 3 parts by volume of phosphoryl chloride at 5 to 10 C. and the resultant mixture is kept standing with stirring at the same temperature for 1.5 hours.

The reaction mixture is poured into a mixture of 15 parts by volume of a 28% aqueous ammonia and parts by volume of ice water and the whole mixture is concentrated under reduced pressure to remove acetone. The concentrated solution is adjusted to pH 4.5 with acetic acid and is allowed to pass through a column packed with 9 parts by weight of activated charcoal. After being washed with water, the column is subjected to elution with 200 parts by volume of a mixture of nbutyl alcohol, ethyl alcohol, a 28% aqueous ammonia and water (5:45 :2:48 by volume ratio). The eluate is concentrated under reduced pressure to give 1.00 part by weight of 2-chloro-9-(2,3-isopropylidene-B-D-ribofuranosyl)hypoxanthine as crystalline powder.

(B) 18 parts by weight of 2-chloro-9-(2,3'-isopropylidene-B-D-ribofuranosyl)hypoxanthine is dissolved in 1000 parts by volume of 2 N sodium ethoxide and the solution is refluxed for 5 hours. The reaction mixture is poured into 1000 parts by volume of ice water. The mixture is adjusted to pH 7 with acetic acid and is then concentrated under reduced pressure to remove ethyl alcohol layer. The resulting aqueous layer is subjected to extraction with 1000 parts by volume of chloroform. The chloroform layer is dehydrated with anhydrous calcium chloride and is then concentrated under reduced pressure to give 20 parts by weight of 2-ethoxy-9-(2',3'-isopropylidene-fl-D-ribofuranosyl)-xanthine as an oily substance (77.5% purity).

Ultraviolet absorption:

new 252 mi When this product is subjected to thin layer chromatography on silica gel employing as the developer a mixture of methyl alcohol and chloroform (1:5), it gives a single ultraviolet absorption figure at Rf 0.5.

(C) 20 parts by weight of 2-ethoxy-9-(2',3'-isopropylidene-fl-D-ribofuranosyl)xanthine of 77.5% purity is cooled at --30 C. and 50 parts by volume of pyrophosphoryl chloride is added thereto dropwise with vigorous stirring. After the addition, the temperature of the resultant mixture is gradually elevated to become 0 C. for 3 hours and the mixture is kept standing with stirring at the same temperature for 3 hours. The reaction mixture is poured into 1000 parts by volume of ice water and the Whole mixture is adjusted to pH 1.5 with a l N sodium hydroxide. The mixture is heated at 70 C. for 15 minutes and then is cooled at room temperature. The mixture is allowed to pass through a column packed with 250 parts by weight of activated charcoal. The column is washed with water and is subjected to elution with 1000 parts by volume of a mixture of ethyl alcohol, n-butyl alcohol, 28% aqueous amonia and water (45 52:48). The eluate is concentrated under reduced pressure to 200 parts by volume. The concentrated solution is mixed with 75 parts by volume of 1 N sodium hydroxide and the mixture is subjected to freeze-drying to give crystalline powder. This is dried on diphosphorus pentoxide at C. for 4 hours to give 13 parts by weight of colorless crystals of sodium 2-ethoxyinosine-5'- phosphate.

The product gives a single ultraviolet absorption figure in paper electrophoresis (0.05 M borate buflfer, pH 9.2), the absorption being nae- 253 i and A9 3 261 m Elementary analysis.Calculated for C H N O PNa H O (percent) N, 12.33; P, 6.83. Found (percent): N, 12.03; P, 7.04.

EXAMPLE 3 Mill 253 u X 103) MEQ' 262 m (6: 117x10 Elementary analysis.Calculated for C H N O PBa- 2H O (percent) C, 27.03; H, 3.66; N, 9.70; P, 5.36. Found (percent): C, 27.17; H, 3.57; N, 9.05; P, 5.20.

This product is treated with 0.23 part by weight of sodium sulfate to give 0.76 part by weight of disodium 2-n-propoxyinosine-5'-phosphate as colorless crystals.

EXAMPLE 4 1 part by Weight of 2-chloro-9-(2',3'-isopropylidene-[3- D-ribofuranosyl)xanthine and 150 parts by volume of 2 N sodium isopropoxide are treated after the manner described in (B) of Example 2 to give 0.98 part by weight of 2 isopropoxy-9-(2,3'-isopropylidene-fi-D-ribofuranosyl)xanthine as powdery crystals.

0.96 part by weight of thus-obtained 2-isopropoxy-9- (2,3'-isopropylidene-B-D-ribofuranosyl)xanthine and 4.5 parts by volume of pyrophosphoryl chloride are treated after the manner described in (C) of *Example 2. to give 0.89 part by weight of barium 2-isopropoxyinosine-5'- phosphate as colorless crystalline powder.

Ultraviolet absorption:

)thlfi' 262 m (6: 11.6 X10 Elemenlary analysis.Calculated for C H N O Ba (percent) I C, 27.03; H, 3.66; N, 9.70; P, 5.36. Found (percent): C, 27.22; H, 4.14; N, 10.01; P, 5.53.

This product is treated with 0.22 part by weight of sodium sulfate to give 0.7 part by weight of disodium 2-isopropoxyinosine-5-phosphate.

EXAMPLE 5 0.07 part of disodium Z-methoxyinosine-5'-phosphate, 10 parts of salt, 5 parts of sugar, 56 part of skimmed milk powder, 3 parts of monosodium glutamate, 4.5 parts of vegetable shortening and 2 parts of onion powder are homogeneously mingled to give about 150 parts by weight of powdery soup composition. 7.0 parts by Weight of the composition is dissolved in 900 parts by volume of hot water to give a soup.

EXAMPLE 6 10 parts by weight of disodium 2-ethoxyinosine-5- phosphate is dissolved in 90 parts by volume of water. The solution is added to 900 parts by Weight of powdered monosodium glutamate and the mixture is kneaded, granulated, dried and sieved to give a granular seasoning composition.

This composition enhances and improves the flavor of, for example, clear soup with its addition thereto at a range of about 0.05 to about 0.1%.

EXAMPLE 7 To 2000 parts by weight of powdered monosodium glutamate there are added a solution of 30 parts by weight of disodium 2-methoxyinosine-5'-phosphate and 50 parts by weight of disodium 5-inosinate in 150 parts by volume of Water. The mixture is kneaded, granulated, dried and sieved to give a granular seasoning composition.

This composition is characterized by high ability of enhancing and improving the flavor of cream soup with its addition thereto at a range of about 0.05 to about 0.1%.

EXAMPLE 8 Raw soy sauce prepared by the conventional method, is sterilized. To 2000 parts by volume of thus-treated soy sauce, there is uniformly added 0.3 part by weight of disodium 2 methoxyinosine 5' phosphate to give soy sauce which has been enhanced in its flavor.

EXAMPLE 9 To 10,000 parts by volume of Worcestershire sauce prepared by the conventional method, thereto is added 1 part by weight of disodium 2-n-propoxyinosine-5'-phosphate and 10 parts by weight of monosodium glutamate to give Worcestershire sauce which has been enhanced in its flavor.

EXAMPLE 10 Tomato juice is prepared by the conventional method. To 10,000 parts by volume of the tomato juice, there are added 120 parts by weight of sugar, 50 parts by weight of sodium chloride, 10 parts by Weight of monosodium glutamate and 2 parts by weight of dipotassium 2-isopropoxyinosine-5'-phosphate and the mixture is sterilized to give tomato juice which has been enhanced in its flavor.

EXAMPLE 11 To 8,000 parts by weight of fish-paste containing of Water, there are added 250 parts by weight of sodium chloride, 30 parts by Weight of monosodium glutamate and 3 parts by weight of disodium 2-ethoxyinosine-5- phosphate and the mixture is kneaded. To the mixture there is added 1000 parts by weight of lard, 350 parts by weight of potato starch and 400 parts by weight of Wheat starch and the whole mingled thoroughly. The resultant product is packed in a casing. Thus-obtained raw fish sausages are boiled at to C. for 1 hour to give fish sausage which has been enhanced in its flavor.

What is claimed is:

1. 2-(lower alkoxy)inosine-5-phosphate, alkoxy having at most 3 carbon atoms.

2. 2-(lower alkoxy)inosine-5'-phosphate according to claim 1, wherein the compound is in the form of an alkali metal, alkaline earth metal or cyclohexylamine salt.

3. A compound according to claim 2, wherein the salt is sodium salt.

4. A compound according to claim 1, wherein the lower alkoxy is methoxy.

5. A compound according to claim 1, wherein the lower alkoxy is ethoxy.

the lower 9 10 6. A compound according to claim 1, wherein the 3,126,372 3/1964 Bannister 260-2115 lower alkoxy is n-propoxy. 3,408,206 10/1968 Yamazaki et a1. 260-211.5

7. A compound according to claim 1, wherein the lower alkoxy is isopropoxy. LEWIS GO'I'I'S, Primary Examiner 5 References Cited J. R. BROWN, Asslstant Examiner UNITED STATES PATENTS US. Cl. X.R. 2,852,506 9/1958 Goldman et a1. 260211.5 99--35, 54, 55, 100 R, 107, 108, 109, 115, 140 N, 144, 3.014.900 2/1961 Schroeder 260-21l.5 10 147 

